Abstract
Modern flow cytometry instruments have become vital tools for high-throughput analysis of single cells. However, as issues with the cellular labeling techniques often used in flow cytometry have become more of a concern, the development of label-free modalities for cellular analysis is increasingly desired. Non-linear optical phenomena (NLO) are of growing interest for label-free analysis because of the ability to measure the intrinsic optical response of biomolecules found in cells. We demonstrate that a light-sheet consisting of a scanned Bessel beam is an optimal excitation geometry for efficiently generating NLO signals in a microfluidic environment. The balance of photon density and cross-sectional area provided by the light-sheet allowed significantly larger two-photon fluorescence intensities to be measured in a model polystyrene microparticle system compared to measurements made using other excitation focal geometries, including a relaxed Gaussian excitation beam often used in conventional flow cytometers.
Highlights
Over the last few decades, flow cytometry has become an invaluable tool in the biomedical field; clinical diagnostics, therapeutics, cell biology, and many other fields benefit from its cellular analysis and sorting capabilities[1,2,3,4,5,6,7,8]
Non-linear optical phenomena or NLO are of growing interest for label-free analysis because of the ability to measure the intrinsic optical response of biomolecules found in cells[12,16,17,18,20,21,22,23,24]
We have previously reported on an intrinsic second harmonic generation signal that could potentially be used as a specific marker for label-free, non-genetic purification of stem cell derived cardiomyocytes.[13]
Summary
Similar to the results with the tightly focused Gaussian beam, scanning of the Bessel beam resulted in lower peak intensities compared to the static Bessel Beam results These results demonstrate that a scanned Bessel beam can be used to achieve a greater photon density and more efficient excitation of NLO than a relaxed Gaussian beam while still providing effective coverage of the cross-sectional area of the sample stream. Effective coverage of the cross-sectional area of the sample stream allowing the entire volume of each particle to be interrogated as was demonstrated with a relaxed Gaussian excitation and a light-sheet system consisting of a scanned Bessel beam Both of these excitation approaches were able to measure well-defined distributions for each particle size, the light-sheet system was able to produce higher peak intensities and showed better distinction between the different distributions due to the higher photon density in the beam. Future, this measurement system will be combined with a sorting mechanism to perform purification of stem cell derived cardiomyocytes through second harmonic generation of light interacting with the myosin domains of the cardiomyocytes
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